Proteins containing non-natural amino acids hold great promise for biomedical and therapeutic purposes. Such amino acids may be particularly useful in the structural and functional probing of proteins, construction of peptide libraries for combinatorial chemistry, and in proteomics. However, the synthesis of such proteins has not heretofore been easy. In the translation system that is known to occur currently in nature, genetic coding is carried out by aminoacyl tRNA synthetases (ARSs). They exist in 20 different forms, each of which specifically catalyzes the esterification of a single amino acid to its cognate tRNA isoacceptor, thereby directly connecting the amino acid with its corresponding anticodon triplet. Because misacylation of noncognate amino acids to tRNAs causes misincorporation of amino acids into cellular proteins which can be fatal to their intracellular activity, the fidelity of the aminoacylation reactions by the ARSs must be extremely high. To achieve this important task, the ARSs use very sophisticated mechanisms to selectively recognize the cognate amino acids and tRNAs. The recognition determinants of tRNAs are diverse ranging from the anticodon loop to the acceptor-Ti˜C stem and the phosphate-ribose backbone. Because of these complexities, engineering of ARSs with desired specificities toward nonnatural tRNAs and amino acids has not been achieved. As a result attention has turned to the nucleic acids.
For many years, nucleic acids were considered to be only informational molecules. However, the pioneering work of Cech and coworkers (Cech, 1987, Science, 236:1532–1539; McCorkle et al., 1987, Concepts Biochem. 64:221–226) demonstrated the presence of naturally occurring RNAs that can act as catalysts (ribozymes). However, although these natural RNA catalysts have only been shown to act on ribonucleic acid substrates for cleavage and splicing, recent development of artificial evolution of ribozymes has expanded the repertoire of catalysis to various chemical reactions. For example, RNAs have been reported to catalyze phosphodiester cleavage on DNA (Beaudry et al., 1992, Science, 257:635), hydrolysis of aminoacyl esters (Piccirilli et al., 1992, Science, 256:1420–1424), self-cleavage (Pan et al., 1992, Biochemistry, 31:3887), ligation of an oligonucleotide with a 3′0H to the 5′ triphosphate end of the catalyst (Bartel et al., 1993, Science, 261:1411–1418), biphenyl isomerase activity (Schultz et al., 1994, Science, 264:1924–1927), and polynucleotide kinase activity (Lorsch et al., 1994, Nature, 371:31–36).
To identify novel catalysts, Brennen et al. (1992, Proc. Natl. Acad. Sci., USA, 89:5381–5383) constructed a heterogenous pool of macromolecules and used an in vitro selection process to isolate molecules that catalyze the desired reaction. A variation of this approach has been used by Gold et al. (U.S. Pat. No. 5,475,096). This method, known as Systematic Evolution of Ligands by Exponential enrichment (SELEX), identifies nucleic acids that have the ability to form specific, non-covalent interactions with a variety of target molecules. A related patent (U.S. Pat. No. 5,990,142) is based on the SELEX method, but can potentially identify modified and non-modified RNA molecules that can catalyze covalent bond formation with a target. Recently, a similar approach was used to identify catalytic RNA molecules having phosphodiesterase, amidase activity (U.S. Pat. No. 6,063,566 to Joyce).
Additionally, studies have identified RNA molecules that can catalyze aminoacyl-RNA bonds on their own (2′)3′-termini. (Illangakekare et al., 1995 Science 267:643–647), or where an RNA molecule can transfer an amino acid from one RNA molecule to another (Lohse et al., 1996, Nature 381:442–444).
However, there has been no demonstration heretofore of catalytic tRNA-like molecules that can cause aminoacylation of RNA molecules which are physiologically significant in modern protein translation processes.